Production of Fatty Acid Alkyl Esters by Use of Two Lipolytic Enzymes

ABSTRACT

A method for producing fatty acid alkyl esters, wherein a solution comprising triglyceride and alcohol is contacted with a first lipolytic enzyme having a relatively higher activity on free fatty acids than on triglyceride and a second lipolytic enzyme having a relatively higher activity on triglyceride than on free fatty acids.

FIELD OF THE INVENTION

The present invention relates to a method for producing fatty acid alkylesters from triglyceride by use of a first lipolytic enzyme whichfavours the conversion of triglyceride to fatty acid alkyl esters and asecond lipolytic enzyme which favours the conversion of free fatty acidsto fatty acid alkyl esters.

BACKGROUND ART

Biodiesel, generally classified as mono-alkyl esters of fats and oils,has become more attractive recently because of its environmentalbenefits. Although biodiesel is at present successfully producedchemically (using e.g. NaOH and/or sodium methoxide as catalyst), thereare several associated problems to restrict its development, such aspre-processing of oil due to high contents of free fatty acids, removalof chemical catalyst from ester and glycerol phase and removal ofinorganic salts during glycerol recovery.

The disadvantages caused by chemical catalysts are largely prevented byusing lipolytic enzymes as the catalysts and in recent years interesthas developed in the use of lipases with or without immobilization intransesterification for the production of biodiesel.

Fungal esterases may be used in the enzymatic production of esters,where they may replace catalysts like mineral acid (e.g. sulphuric acid,hydrogen chloride, and chlorosulfonic acid), amphoteric hydroxides ofmetals of groups I, II, III, and IV, and others. The use of enzymes forester synthesis has been described in the prior art, in particularenzymes classified in EC 3.1.1 Carboxylic ester hydrolases according toEnzyme Nomenclature (Recommendations of the Nomenclature Committee ofthe International Union of Biochemistry and Molecular Biology, 1992 orlater).

WO 88/02775 discloses lipases A and B from Candida antarctica. It statesthat C. antarctica lipase B (CALB) is more effective for estersynthesis.

Cutinases are lipolytic enzymes capable of hydrolyzing the substratecutin. Cutinases are known from various fungi (P. E. Kolattukudy in“Lipases”, Ed. B. Borgström and H. L. Brockman, Elsevier 1984, 471-504).The amino acid sequence of a cutinase from Humicola insolens has beenpublished (U.S. Pat. No. 5,827,719).

Many researchers have reported that a high yield of alkyl esters couldbe reached in the presence of organic solvents, but because of thetoxicity and flammability of organic solvents lipase-catalysedalcoholysis in a solvent-free medium is more desirable. Methanolysiscatalysed by lipases has been shown to take place in a water-containingsystem free of organic solvents. In such systems lipases which are lesssensitive to methanol is advantageous (Kaieda et al. J. Biosci. Bioeng.2001, 91:12-15). It is well known that excessive short-chain alcoholssuch as methanol might inactivate lipase seriously. However, at leastthree molar equivalents of methanol are required for the completeconversion of the oil to its corresponding methyl ester. Du et al.(Biotechnol. Appl. Biochem. 2003, 38:103-106) studied the effect ofmolar ratio of oil/methanol comparatively during non-continuous batchand continuous batch operation.

To avoid inactivation of the lipases the methanol concentration has beenkept low by step-wise addition of methanol throughout the reaction(Shimada et al. J Mol. Catalysis Enzymatic, 2002, 17:133-142; Xu et al.2004, Biocat. Biotransform. 22:45-48).

Boutur et al. (J. Biotechnol. 1995, 42:23-33) reported a lipase fromCandida deformans which were able to catalyse both alcoholysis oftriglyceride (TG) and esterification of free fatty acids (FFA), but notunder the same reaction conditions. Under the conditions described byBoutur et al. only the esterification was catalysed.

In order to obtain a more economic production of fatty acid ethyl estersfor biodiesel, there is a need for a faster conversion of fats and oilsto their corresponding methyl esters and a higher yield in saidconversion.

SUMMARY OF THE INVENTION

The present invention relates to a method for producing fatty acid alkylesters, such as fatty acid methyl esters (FAME) and fatty acid ethylesters. Such esters are also called biodiesel, because they are used asan additive to mineral diesel to result in a sulphur-free,higher-cetane-number fuel, which is partly based on renewable resources.

The method of the invention includes a solution comprising alcohol,triglyceride and/or free fatty acids, which solution is contacted with afirst lipolytic enzyme and a second lipolytic enzyme of differentspecificity, wherein the lipolytic enzymes catalyse the conversion oftriglyceride or free fatty acids or a mixture of both to fatty acidalkyl esters. The first lipolytic enzyme is characterised in that itexhibits higher activity against triglyceride than free fatty acids,whereas the second lipolytic enzyme exhibits higher activity againstfree fatty acids than triglyceride. The activity of the first and secondlipolytic enzymes is determined by use of the methods described inExample 1 and 2 below.

The first lipolytic enzyme is defined as one having a ratio of activityon TG/activity on FFA below 0.2. The second lipolytic enzyme is definedas one having a ratio of activity on TG/activity on FFA above 0.5.

The combination of a first lipolytic enzyme and a second lipolyticenzyme according to the present invention results in a synergisticeffect on the conversion of triglyceride and triglyceride in combinationwith free fatty acids to fatty acid alkyl esters, whereby a higherpercentage of conversion is obtained in a shorter period of time.

Further, the invention relates to a batch process or a continuous,staged process to produce fatty acid alkyl esters using a first and asecond lipolytic enzyme as described above, wherein the alcohol is addedcontinuously or stepwise, and wherein the enzymes are recycled or usedonly once.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for producing fatty acid alkylesters. The method of the invention includes a solution comprisingalcohol, and a substrate, which comprises triglyceride and/or free fattyacids. The solution is contacted with a first lipolytic enzyme and asecond lipolytic enzyme of different specificity, wherein the lipolyticenzymes catalyse the conversion of triglyceride or free fatty acids or amixture of both to fatty acid alkyl esters.

Substrates Suitable substrates for production of fatty acid alkyl estersin accordance with the present invention are a broad variety ofvegetable oils and fats; rapeseed and soybean oils are most commonlyused, though other crops such as mustard, sunflower, canola, coconut,hemp, palm oil and even algae show promise. The substrate can be ofcrude quality or further processed (refined, bleached and deodorized).Also animal fats including tallow, lard, poultry, marine oil as well aswaste vegetable and animal fats and oil, commonly known as yellow andbrown grease can be used. The suitable fats and oils may be puretriglyceride or a mixture of triglyceride and free fatty acids, commonlyseen in waste vegetable oil and animal fats. The substrate may also beobtained from vegetable oil deodorizer distillates. The type of fattyacids in the substrate comprises those naturally occurring as glyceridesin vegetable and animal fats and oils. These include oleic acid,linoleic acid, linolenic acid, palmetic acid and lauric acid to name afew. Minor constituents in crude vegetable oils are typicallyphospholipids, free fatty acids and partial glycerides i.e. mono- anddiglycerides. When used herein the phrase “fatty acid residues” refersto fatty acids, either free or esterified as in triglycerides,diglycerides, monoglycerides or fatty acid alkyl esters.

Biodiesel Fatty acid alkyl esters, such as fatty acid methyl esters(FAME) and fatty acid ethyl esters are also called biodiesel, becausethey are used as an additive to fossil diesel. Biodiesel constitutes anincreasingly important additive or substitute for diesel fuels based onfossil oil because it is produced from renewable resources.

Alcohol The alcohol used in the method of the invention is preferably alower alcohol having 1 to 5 carbon atoms (C₁-C₅). Preferred alcohols aremethanol and ethanol.

Lipolytic enzyme The first lipolytic enzyme according to the presentinvention is characterised in that it exhibits higher activity againsttriglyceride than free fatty acids, whereas the second lipolytic enzymeexhibits higher activity against free fatty acids than triglyceride. Theactivity of the lipolytic enzymes against triglycerides and free fattyacid is determined as described in Example 1 and Example 2,respectively.

According to the present invention, the first lipolytic enzyme isdefined as one having a ratio of activity on triglyceride (measured asconversion of triglyceride to fatty acid alkyl esters) to activity onFFA (measured as conversion of FFA to fatty acid alkyl esters) below0.2. The second lipolytic enzyme is defined as one having a ratio ofactivity on triglyceride (measured as conversion of triglyceride tofatty acid alkyl esters) to activity on FFA (measured as conversion ofFFA to fatty acid alkyl esters) above 0.5.

Accordingly, the present invention relates to a method for producingfatty acid alkyl esters, characterised in that a solution comprisingtriglyceride and alcohol is contacted with a first lipolytic enzymehaving a ratio of activity on triglyceride to activity on FFA below 0.2and a second lipolytic enzyme having a ratio of activity on triglycerideto activity on FFA above 0.5.

The first lipolytic enzyme preferably has a ratio of activity ontriglyceride to activity on FFA in the range of 0.01-0.2, morepreferably in the range of 0.01-0.1, more preferably in the range of0.0125-0.05, more preferably in the range of 0.015-0.025, even morepreferably in the range of 0.02-0.024. The second lipolytic enzymepreferably has a ratio of activity on triglyceride to activity on FFA inthe range of 0.5-20, more preferably in the range of 0.6-10, morepreferably in the range of 0.7-5, even more preferably in the range of0.8-1.5.

As stated above, the activity of the lipolytic enzymes againsttriglycerides and free fatty acid is determined as described in Example1 and Example 2, respectively. Below, the ratio of activity ontriglyceride (abbreviated TG) as measured in Example 1 to activity onfree fatty acids (abbreviated FFA) as measured in Example 2, has beencalculated for the tested lipolytic enzymes:

CALB: TG/FFA=0.55/26.41=0.021

H. insolens cutinase: TG/FFA=12.13/10=1.213

T. lanuginosus lipase: TG/FFA=13.22/16.25=0.814.

The combination of a first lipolytic enzyme and a second lipolyticenzyme according to the present invention results in a synergisticeffect on the conversion of triglyceride and/or free fatty acids tofatty acid alkyl esters, whereby a higher percentage of conversion isobtained in a shorter period of time.

In a preferred embodiment of the method of the present invention a firstlipolytic enzyme of the present invention is lipase B from Candidaantarctica (CALB) as disclosed in WO 88/02775, whereas the secondlipolytic enzyme is one of the Thermomyces lanuginosus (previouslyHumicola lanuginosus) lipase variants exemplified in WO 00/60063 and theHumicola insolens cutinase variants disclosed in Example 2 of WO01/92502, hereinafter referred to as T. lanuginosus lipase and H.insolens cutinase respectively. In a second preferred embodiment a firstlipolytic enzyme includes Hyphozyma sp. lipase and Candida parapsilosislipase, whereas a second lipolytic enzyme of the present inventionincludes C. antarctica lipase A as disclosed in WO 88/02775 and lipasesfrom Humicola lanuginosus (EP 258 068), Candida rugosa, Pseudomonascepacia, Geotricum candidum, Rhizomucor miehei, Ctytococcus spp. S-2 andCandida parapsilosis.

In a third embodiment the first lipolytic enzyme is homologous withCALB, Hyphozyma sp. lipase or Candida parapsilosis lipase, whereas thesecond lipolytic enzyme is homologous with T. lanuginosus lipase, H.insolens cutinase or any of the lipases from Humicola lanuginosus (EP258 068), Candida rugosa, Pseudomonas cepacia, Geotricum candidum,Rhizomucor miehei, Crytococcus spp. S-2 and Candida parapsilosis.

Preferably, the first lipolytic enzyme according to the method of thepresent invention is 60% identical with CALB, whereas the secondlipolytic enzyme is 60% identical with the T. lanuginosus lipase, the H.insolens cutinase. More preferably the first lipolytic enzyme is 70%identical with CALB, even more preferably the first lipolytic enzyme is75%, 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or even 99%identical with CALB. Similarly, the second lipolytic enzyme ispreferably 70% identical with T. lanuginosus lipase and H. insolenscutinase, more preferably the second lipolytic enzyme is 75%, 80%, 85%,88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or even 99% identical with T.lanuginosus lipase or H. insolens cutinase.

The enzymes may be applied as lyophilised powder, immobilised or inaqueous solution.

For purposes of the present invention, the degree of identity may besuitably determined according to the method described in Needleman, S.B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-45,with the following settings for polypeptide sequence comparison: GAPcreation penalty of 3.0 and GAP extension penalty of 0.1. Thedetermination may be done by means of a computer program known such asGAP provided in the GCG program package (Program Manual for theWisconsin Package, Version 8, August 1994, Genetics Computer Group, 575Science Drive, Madison, Wis., USA 53711).

Two given sequences can be aligned according to the method described inNeedleman (supra) using the same parameters. This may be done by meansof the GAP program (supra).

Further, the invention relates to a batch process and/or a continuous,staged process to produce fatty acid alkyl esters using a first and asecond lipolytic enzyme as described above, wherein the alcohol is addedcontinuously or stepwise, and wherein the enzymes are recycled or usedonly once. If the enzymes are in an aqueous phase, this phase can beseparated from the fatty phase by a decanter, a settler or bycentrifugation. In the continuously process the two phases, oil andaqueous, respectively, can be processed counter-currently. Kosugi, Y;Tanaka, H. and Tomizuka, (1990), Biotechnology and Bioengineering, vol.36, 617-622, describes a continuous, counter-current process tohydrolyse vegetable oil by immobilized lipase.

General Description of Preparation of Fatty Acid Alkyl Esters

The substrate comprising triglyceride is mixed with alcohol, preferablymethanol or ethanol and heated to 30-60° C., preferably 50° C. on areciprocal water shaking bath (200 rpm). Preferably water is added andthe solution is mixed and further heated to the desired temperature. Theenzymes are added and the solution is mixed vigorously and left onreciprocal water shaking bath at the desired temperature, preferably 50°C. and 200 rpm to react. The phases of the reaction mixture can be mixedby the use of high shear mixers, such as types from Silverson or IKALabortechnik, as used in enzymatic degumming of vegetable oil (Clausen,K. (2001), European Journal of Lipid Science and Technology, vol. 103,333-340).

The [methanol]/[fatty acid residue] molar ratio should be at least 0.1and maximum 10, preferable in the range 0.3-5, more preferable 0.4-2.The alcohol can be added stepwise to the reaction over time. Water canbe added separately or within an aqueous enzyme solution. The finalconcentration of water in the reaction mixture can be 0-50% (w/w),preferably 5-40%, more preferably 5-30%. The substrate comprises 1-99%(w/w) triglyceride, preferably in the range of 70-95%. Further, thesubstrate may comprise free fatty acids amounting to 0.01-95% (w/w),preferably in the range of 0.01-30%. Also, mono- and diglycerides andphospholipids may be present.

The course of the reaction can be followed by withdrawing samples fromthe reaction mixture after a certain period of reaction time. Thesamples are centrifuged for 14 minutes at 14000 rpm. The upper layerconsists of fatty material not soluble in the water phase and this isanalyzed by ¹H NMR (using CDCl₃ as solvent). After the reaction hasended the glycerol phase is removed either by decanting orcentrifugation.

Cloning a DNA Sequence Encoding a Lipolytic Enzyme

The DNA sequence encoding a parent lipolytic enzyme may be isolated fromany cell or microorganism producing the lipolytic enzyme in question,using various methods well known in the art. First, a genomic DNA and/orcDNA library should be constructed using chromosomal DNA or messengerRNA from the organism that produces the lipolytic enzyme to be studied.Then, if the amino acid sequence of the lipolytic enzyme is known,labelled oligonucleotide probes may be synthesized and used to identifylipolytic enzyme-encoding clones from a genomic library prepared fromthe organism in question. Alternatively, a labelled oligonucleotideprobe containing sequences homologous to another known lipolytic enzymegene could be used as a probe to identify lipolytic enzyme-encodingclones, using hybridization and washing conditions of lower stringency.

Yet another method for identifying lipolytic enzyme-encoding cloneswould involve inserting fragments of genomic DNA into an expressionvector, such as a plasmid, transforming cutinase-negative bacteria withthe resulting genomic DNA library, and then plating the transformedbacteria onto agar containing a substrate for lipolytic enzyme (i.e.triglyceride), thereby allowing clones expressing the lipolytic enzymeto be identified.

Alternatively, the DNA sequence encoding the enzyme may be preparedsynthetically by established standard methods, e.g. the phosphoroamiditemethod described by S. L. Beaucage and M. H. Caruthers, (1981),Tetrahedron Letters 22, p. 1859-1869, or the method described by Mattheset al., (1984), EMBO J. 3, p. 801-805. In the phosphoroamidite method,oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer,purified, annealed, ligated and cloned in appropriate vectors.

Finally, the DNA sequence may be of mixed genomic and synthetic origin,mixed synthetic and cDNA origin or mixed genomic and cDNA origin,prepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate, the fragments corresponding to various parts of the entireDNA sequence), in accordance with standard techniques. The DNA sequencemay also be prepared by polymerase chain reaction (PCR) using specificprimers, for instance as described in U.S. Pat. No. 4,683,202 or R. K.Saiki et al., (1988), Science 239, 1988, pp. 487-491.

Expression Vector

The recombinant expression vector carrying the DNA sequence encoding alipolytic enzyme of the invention may be any vector which mayconveniently be subjected to recombinant DNA procedures, and the choiceof vector will often depend on the host cell into which it is to beintroduced. The vector may be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated. Examples ofsuitable expression vectors include pMT838.

The expression vector of the invention may also comprise a suitabletranscription terminator and, in eukaryotes, polyadenylation sequencesoperably connected to the DNA sequence encoding the lipolytic enzyme ofthe invention. Termination and polyadenylation sequences may suitably bederived from the same sources as the promoter.

The vector may further comprise a DNA sequence enabling the vector toreplicate in the host cell in question. Examples of such sequences arethe origins of replication of plasmids pUC19, pACYC177, pUB110, pE194,pAMB1 and pIJ702.

The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, such as the dalgenes from B. subtilis or B. licheniformis, or one which confersantibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracyclin resistance. Furthermore, the vector may comprise Aspergillusselection markers such as amdS, argB, niaD and sC, a marker giving riseto hygromycin resistance, or the selection may be accomplished byco-transformation, e.g. as described in WO 91/17243.

The procedures used to ligate the DNA construct of the inventionencoding a cutinase variant, the promoter, terminator and otherelements, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor,1989).

Promoter

In the vector, the DNA sequence should be operably connected to asuitable promoter sequence. The promoter may be any DNA sequence whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell.

Examples of suitable promoters for directing the transcription of theDNA sequence encoding a lipolytic enzyme of the invention, especially ina bacterial host, are the promoter of the lac operon of E. coli, theStreptomyces coelicolor agarase gene dagA promoters, the promoters ofthe Bacillus licheniformis alfa-amylase gene (amyL), the promoters ofthe Bacillus stearothermophilus maltogenic amylase gene (amyM), thepromoters of the Bacillus amyloliquefaciens alfa-amylase (amyQ), thepromoters of the Bacillus subtilis xylA and xylB genes etc. Fortranscription in a fungal host, examples of useful promoters are thosederived from the gene encoding A. oryzae TAKA amylase, the TPI (triosephosphate isomerase) promoter from S. cerevisiae (Alber et al. (1982),J. Mol. Appl. Genet 1, p. 419-434, Rhizomucor miehei asparticproteinase, A. niger neutral alfa-amylase, A. niger acid stablealfa-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzaealkaline protease, A. oryzae triose phosphate isomerase, or A. nidulansacetamidase.

Host Cells

The cell of the invention, either comprising a DNA construct or anexpression vector of the invention as defined above, is advantageouslyused as a host cell in the recombinant production of a lipolytic enzymeof the invention. The cell may be transformed with the DNA construct ofthe invention encoding the lipolytic enzyme, conveniently by integratingthe DNA construct (in one or more copies) in the host chromosome. Thisintegration is generally considered to be an advantage as the DNAsequence is more likely to be stably maintained in the cell. Integrationof the DNA constructs into the host chromosome may be performedaccording to conventional methods, e.g. by homologous or heterologousrecombination. Alternatively, the cell may be trans-formed with anexpression vector as described above in connection with the differenttypes of host cells.

The cell of the invention may be a cell of a higher organism such as amammal or an insect, particularly a microbial cell, e.g. a bacterial ora fungal (including yeast) cell.

Examples of suitable bacteria are Gram positive bacteria such asBacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillusbrevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyceslividans or Streptomyces murinus, or gram negative bacteria such as E.coli. The transformation of the bacteria may, for instance, be effectedby protoplast transformation or by using competent cells in a mannerknown per se.

The yeast organism may favorably be selected from a species ofSaccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae.

The host cell may also be a filamentous fungus e.g. a strain belongingto a species of Aspergillus, particularly Aspergillus oryzae orAspergillus niger, or a strain of Fusarium, such as a strain of Fusariumoxysporum, Fusarium graminearum (in the perfect state named Gibberellazeae, previously Sphaeria zeae, synonym with Gibberella roseum andGibberella roseum f. sp. cerealis), or Fusarium sulphureum (in theprefect state named Gibberella puricaris, synonym with Fusariumtrichothecioides, Fusarium bactridioides, Fusarium sambucinum, Fusariumroseum, and Fusarium roseum var. graminearum), Fusarium cerealis(synonym with Fusarium crokkwellense), or Fusarium venenatum.

In a particular embodiment of the invention the host cell is a proteasedeficient or protease minus strain. This may for instance be theprotease deficient strain Aspergillus oryzae JaL 125 having the alkalineprotease gene named “alp” deleted. This strain is described in WO97/35956 (Novo Nordisk).

Filamentous fungi cells may be transformed by a process involvingprotoplast formation and transformation of the protoplasts followed byregeneration of the cell wall in a manner known per se. The use ofAspergillus as a host microorganism is described in EP 238 023 (NovoNordisk A/S), the contents of which are hereby incorporated byreference.

Production of Lipolytic Enzyme by Cultivation of Transformant

The invention relates, inter alia, to a method of producing a lipolyticenzyme of the invention, which method comprises cultivating a host cellunder conditions conducive to the production of the lipolytic enzyme andrecovering the lipolytic enzyme from the cells and/or culture medium.

The medium used to cultivate the cells may be any conventional mediumsuitable for growing the host cell in question and obtaining expressionof the lipolytic enzyme of the invention. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedrecipes (e.g. as described in catalogues of the American Type CultureCollection).

The lipolytic enzyme secreted from the host cells may conveniently berecovered from the culture medium by well-known procedures, includingseparating the cells from the medium by centrifugation or filtration,and precipitating proteinaceous components of the medium by means of asalt such as ammonium sulphate, followed by the use of chromatographicprocedures such as ion exchange chromatography, affinity chromatography,or the like.

Materials and Methods Lipase Activity on Tributyrin (LU)

A substrate for lipolytic enzymes is prepared by emulsifying tributyrin(glycerin tributyrate) using gum Arabic as emulsifier. The hydrolysis oftributyrin at 30° C. at pH 7 is followed in a pH-stat titrationexperiment. One unit of lipase activity (1 LU) equals the amount ofenzyme capable of releasing 1 μmol butyric acidimin at the standardconditions.

Preparation of Fatty Acid Alkyl Ester

8.00 gram of substrate is mixed with methanol (0.500 ml=>0.395 gram).The following types of substrates were used:

-   Example 1) 100% salad oil (refined, bleached and deodorized soybean    oil, RBD SBO);-   Example 2) 100% oleic acid;-   Example 3) mixture of 20% w/w oleic acid in RBD SBO

The substrate-methanol mixture is heated to 50° C. on a reciprocal watershaking bath (200 rpm). Demineralised water is added (volume dependingon added enzyme volume; total amount of water: 4.00 ml including waterfrom enzyme addition), corresponding to 32 w/w % of the total mixture.The mixture is heated to 50° C. Then enzyme is added to the mixture andvigorously mixed for 10 sec. and left on reciprocal water shaking bathat 50° C. and 200 rpm. The phases of the reaction mixture can be mixedby the use of high shear mixers, such as types from Silverson Ltd. UK orIKA Kunkel.

Samples are withdrawn from the reaction mixture after 3 hrs. reactiontime and centrifuged for 14 minutes at 14000 rpm. The upper layerconsists of fatty material not soluble in the water phase and this isanalyzed by ¹H NMR (using CDCl₃ as solvent) Varian 400 MHz spectrometer(Varian Inc. CA, USA). The conversion of the fatty acids residues intofatty acid methyl ester is determined by the ratio of the methyl signalsfrom the fatty acid methyl esters, —COOCH₃ (3.70 ppm) and CH₃CH₂—(1.0-0.9 ppm) from the fatty acid residues.

The enzyme dose is based on a total 0.4 mg protein/8.00 gram substrate.For testing a synergistic effect of two enzymes combined; 0.2 mg of eachenzyme were added to 8 gram of substrate and compared to the each of thesingle enzymes at a dose of 0.4 mg/8 gram of substrate. To relate theamount of protein to an enzyme activity, a standard enzyme activityassay can be applied, in this case the LU-assay as described above(lipase activity on tributyrine). The following enzyme preparations weretested:

1. T. lanuginosus lipase (TLL, specific activity 7000 LU/mg protein)

2. C. antarctica lipase B (CALB, specific activity 500 LU/mg protein)

3. H. insolens cutinase (cutinase, specific activity 1800 LU/mg protein)

Enzyme dose and additional water volumes for experiments with singleenzymes:

1. TLL: 0.700 ml of a 4000 LU/ml enzyme solution+3.30 ml water

2. CALB: 1.680 ml of a 119 LU/ml enzyme solution+2.32 ml water

3. Cutinase: 0.450 ml of a 1600 LU/ml enzyme solution+3.55 ml water

Enzyme dose and additional water volumes for experiments withcombination of enzymes:

1. TLL+CALB: (0.350 ml of a 4000 LU/ml TLL solution+0.840 ml of a 119LU/ml CALB solution+2.810 ml water)

2. Cutinase+CALB: (0.225 ml of a 1600 LU/ml cutinase solution+0.840 mlof a 119 LU/ml CALB solution+2.935 ml water).

EXAMPLES Example 1 Preparation of Fatty Acid Alkyl Esters fromTriglycerides

Refined, bleached and deodorized soybean oil (RBD SBO, salad oil) wasused as substrate according to the general method described above.

The conversions (%) of fatty acid residues into FAME after 3 hoursreaction time using different lipolytic enzymes are shown in Table 1,whereas the conversion (%) achieved with a combination of CALB and TLLis shown in Table 2. Coefficient of Variation in % (CV %) of fouridentical experiments was determined to be 2.2%.

TABLE 1 Single enzymes, % conversion of fatty acid residues into FAMECALB 0.55 Cutinase 12.13 TLL 13.22

TABLE 2 Combination of enzymes, % conversion of fatty acid residues intoFAME. CALB + TLL 16.21

Example 2 Preparation of Fatty Acid Alkyl Esters from Oleic Acid

Oleic acid was used as substrate according to the general methoddescribed above. The conversions (%) of fatty acid residues into FAMEafter 3 hours reaction time using different lipolytic enzymes are shownin Table 3.

TABLE 3 Single enzymes, % conversion of fatty acid residues into FAME.CALB 26.41 Cutinase 10 TLL 16.25

Example 3 Preparation of Fatty Acid Alkyl Esters from TriglycerideContaining Free Fatty Acids

A mixture of 20% w/w oleic acid in RBD SBO was used as substrateaccording to the general method described above. The conversions (%) offatty acid residues into FAME after 3 hours reaction time usingdifferent lipolytic enzymes and combinations of said enzymes are shownin Table 4 and 5.

TABLE 4 Single enzymes, % conversion of fatty acid residues into FAME.CALB 16.58 Cutinase 11.22 TLL 14.09

TABLE 5 Combination of enzymes, % conversion of fatty acid residues intoFAME. CALB + Cutinase 18.82 CALB + TLL 20

1-15. (canceled)
 16. A method for producing fatty acid alkyl esters,comprising making a reaction mixture by contacting a solution comprisinga substrate and an alcohol, which substrate comprises triglyceride, witha first lipolytic enzyme having a ratio of activity on triglyceride toactivity on FFA below 0.2 and a second lipolytic enzyme having a ratioof activity on triglyceride to activity on FFA above 0.5.
 17. The methodof claim 16, wherein the substrate further comprises free fatty acids inthe range of 0.01-95% per weight free fatty acids.
 18. The method ofclaim 16, wherein the triglyceride is derived from one or more ofvegetable oil feedstock, rapeseed oil, soybean oil, mustard oil,sunflower oil, canola oil, coconut oil, hemp oil, palm oil, tall oil,animal fats including tallow, lard, poultry and fish oil.
 19. The methodof claim 16, wherein the molar ratio between alcohol and fatty acidresidues is least 0.1 and maximum
 10. 20. The method of claim 16,wherein the molar ratio between alcohol and fatty acid residues is inthe range 0.3-5.
 21. The method of claim 16, wherein the molar ratiobetween alcohol and fatty acid residues is in the range 0.4-2.
 22. Themethod of claim 16, wherein the alcohol is methanol or ethanol.
 23. Themethod of claim 16, wherein the reaction mixture further compriseswater.
 24. The method of claim 16, wherein the first lipolytic enzymehas a ratio of activity on triglyceride to activity on FFA in the rangeof 0.01-0.2 and the second lipolytic enzyme has a ratio of activity ontriglyceride to activity on FFA in the range of 0.5-20.
 25. The methodof claim 16, wherein the first lipolytic enzyme is 60% identical with alipolytic enzyme selected from the group consisting of Lipase B fromCandida antarctica, Hyphozyma sp. lipase and Candida parapsilosislipase.
 26. The method of claim 235 wherein the first lipolytic enzymeis Lipase B from Candida antarctica, Hyphozyma sp. lipase or Candidaparapsilosis lipase.
 27. The method of claim 16, wherein the secondlipolytic enzyme is 60% identical with a lipolytic enzyme selected fromthe group consisting of T. lanuginosus lipase, H. insolens cutinase, C.antarctica lipase A, lipases from Candida rugosa, Pseudomonas cepacia,Geotricum candidum, Rhizomucor miehei, Crytococcus spp. S-2, Candidaparapsilosis and Humicola lanuginosus.
 28. The method of claim 27,wherein the second lipolytic enzyme is one of T. lanuginosus lipase, H.insolens cutinase, C. antarctica lipase A, lipases from Candida rugosa,Pseudomonas cepacia, Geotricum candidum, Rhizomucor miehei, Cytococcusspp. S-2, Candida parapsilosis and Humicola lanuginosus.
 29. The methodof claim 16, wherein the process is proceeding in a batch mode.
 30. Themethod of claim 16, wherein the process is proceeding in a continuousmode.
 31. The method of claim 16, further comprising mixing solutionphases in the reaction mixture using a high shear mixer.
 32. The methodof claim 16, wherein the process is conducted in a counter-current mode.